The present invention relates to methods of testing insects for resistance to pesticides, and in particular to rapid bioassay methods for testing insect resistance to Bacillus thuringiensis (Bt), spinosyns (e.g., spinosad), and pyrethroid insecticides. The present invention further relates to assays for the identification of insect species based on resistance or susceptibility to insecticides, and in particular to a method of distinguishing larvae of Helicoverpa zea and Heliothis virescens. The present invention further relates to methods of screening compounds for insecticidal activity. The present invention further relates to dehydrated insect meal pads and containers for carrying out the inventive methods.
The bacterium Bacillus thuringiensis (Bt) contains genes encoding insecticidal proteins. Bt proteins are toxic when ingested by susceptible insect and insect larvae. Bt proteins are used commercially in pesticide formulations, and transgenic crop plants expressing the Bt gene are widely cultivated. The Bt gene codes for a protein toxin that attacks the insect midgut, stops feeding and eventually kills susceptible insects. Gill et al., Annu. Rev. Entomol. 37:615 (1992); Fischhoff, In Biotechnology and Integrated Pest Management, Ed. G J Persley, pp. 214-227, CAB International, Cambridge, UK.
Several hundred strains of Bacillus thuringiensis exist, with considerable specificity toward various groups of insects such as the lepidoptera (butterflies and moths), coleoptera (beetles) and/or diptera (mosquitoes), as well as toward nematodes. There is a species specificity of the interaction between Bt toxin and the membranes of insect gut cells. The Bt toxin of a particular B. thuringiensis strain may bind to the gut of lepidopteran larvae, or only some species of lepidopteran larvae, but not to others. Binding of the protein to the membrane is required for its toxic effects. Thus the Bt toxins have a high specificity for a small number of pest species, while having no significant activity against beneficial insects, wildlife or humans. Lambert and Peferoen, BioScience, 42:112 (1992); Gill et al., Annu. Rev. Entomol. 37:615 (1992); Meadows, In: Bacillus thuringiensis, An Environmental Biopesticide: Theory and Practice, Entwistle et al., Eds., pp. 193-200 (1993).
Formulations of Bt toxin for use as insecticides are known in the art. See, e.g., U.S. Pat. No. 5,747,450; U.S. Pat. No. 5,250,515; U.S. Pat. No. 5,024,837; U.S. Pat. No. 4,797,276; and U.S. Pat. No. 4,713,241.
Plants transformed to carry the Bt gene and express insecticidal proteins are known in the art, and include potato, cotton, tomato, corn, tobacco, lettuce and canola. Krimsky and Wrubel, Agricultural Biotechnology: An Environmental Outlook, Tufts University, Department of Urban and Environmental Policy, p. 29 (1993). See also U.S. Pat. No. 5,608,142; U.S. Pat. No. 5,495,071; U.S. Pat. No. 5,349,124; and U.S. Pat. No. 5,254,799. The use of such genetically engineered plants is expected to reduce the use of broad spectrum insecticides. Gasser and Fraley, Science 244:1293 (1989).
The use of pesticides results in the selection of individuals resistant to the pesticide, and can lead to the development of pesticide-resistant populations. Resistance to chemical insecticides such as organochlorines, organophosphates, carbamates, spinosyns and pyrethroids is known. Laboratory and field evidence documents that many pests are capable of evolving high levels of resistance to a number of commonly used Bt toxins. Tabashnik, Annu. Rev. Entomol. 39:47 (1994); Tabashnik, J. Econ. Entomol. 83:1671 (1990); Bauer, Fla. Ent. 78:414 (1995); Gould, Proc. Natl. Acad Sci. USA 94:3519 (1997). Resistance may evolve whether the Bt is applied to plants or the plants are genetically engineered to express Bt. The development of resistance to Bt toxin-expressing crops may also result in resistance to commercial formulations of fermented strains of Bt, such as DIPEL(copyright) (Abbott Laboratories).
A further concern in the use of plants genetically engineered to express Bt toxins is the difficulty of distinguishing between different pest species that will and will not be controlled by Bt. The presence of a pest in the field that is resistant to Bt indicates the need for supplemental pesticide treatments, whereas no additional treatment is needed if pests are susceptible to Bt. In the case of cotton, transgenic Bt cultivars are exceptionally toxic to most strains of the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) (Jenkins et al., J. Econ. Entomol. 86:181 (1993)), but are less toxic to the bollworm Helicoverpa zea (Boddie)(Lepidoptera:Noctuidae) (Lambert et al., In: Proceedings Beltwide Cotton Conference, pp. 931-935, National Cotton Council, Memphis, Tenn. (1996)). H. zea and H. virescens are found in the same geographic areas, and in years when H. zea populations are high, larva that are not killed by ingestion of Bt can cause significant damage to cotton. The eggs and young larvae of H. zea and H. virescens are indistinguishable by simple observation in the field (although adults are readily distinguished visually). Without a test to distinguish among susceptible and relatively more tolerant species, farmers finding lepidopteran eggs or neonates on cotton cannot rely on Bt cotton for control of lepidopteran pests.
Rapid, reliable methods to distinguish Bt-susceptible from Bt-resistant species, and to detect the development of Bt resistance, as well as resistance to other insecticides, in populations of insects, are desirable. The methods of the present invention provide a bioassay capable of distinguishing between H. virescens and H. zea. The present methods can also detect insect resistance to Bt, and other insecticides, within a species. The present invention further provides a bioassay for screening compounds to identify those with insecticidal activity.
Accordingly, one aspect of the present invention is a dehydrated insect meal pad comprising a gel matrix and insect meal. In preferred embodiments, the gel matrix comprises agar. In particular preferred embodiments, the meal pad further comprises an insecticide or a compound to be screened for insecticidal activity and a marker compound (e.g., Trypan Blue). Preferred insecticides include Bt toxin and spinosyns.
As a further aspect, the present invention provides a container for housing insects, comprising: (a) a chamber having a floor, sidewalls extending from said floor and an open end portion; (b) a seal member removably attached to the container and configured to close the open end portion; and (c) a dehydrated insect meal pad contained with the chamber, as described above. In preferred embodiments, the floor of the container is permeable to liquids.
As a further aspect, the present invention provides a kit for testing insects for resistance to an insecticide that causes feeding disruption, comprising: (a) one or more containers sized to house at least one of the insects; (b) a dehydrated meal pad comprising a test diet comprising the insecticide; (c) printed instructions setting forth (i) a method for rehydrating the meal pad prior to use, and (ii) a diagnostic time period and a diagnostic amount of feces that indicates the insects are resistant to the insecticide to be tested.
Another aspect of the invention is a kit for screening a test compound for insecticidal activity as indicated by feeding disruption, comprising: (a) one or more containers sized to house at least one insect; (b) a dehydrated meal pad comprising a test diet comprising the test compound; (c) printed instructions setting forth a method for (i) rehydrating the meal pad prior to use, and a diagnostic time period and (ii) a diagnostic amount of feces that indicates the test compound exhibits insecticidal activity.
As still a further aspect the present invention provides a method of detecting in a plurality of insect larvae with the visual appearance of H. virescens larvae, the presence of H. zea larvae, comprising: (a) giving each of the larvae access to an insect meal pad of the invention for a predetermined time, wherein the meal pad has been rehydrated and further wherein the meal pad comprises a test diet containing a predetermined diagnostic amount of Bacillus thuringiensis toxin, and (b) assessing the amount of feces produced by each of the larvae over said predetermined time, wherein any larva producing more than a predetermined diagnostic amount of feces are H. zea. 
As another aspect, the present invention provides a method of detecting, in one or more insects, the presence of an insect(s) resistant to an insecticide that causes feeding disruption in susceptible insects, comprising: (a) giving the one or more insects access to a meal pad according to the invention for a predetermined time, wherein the meal pad has been rehydrated and further wherein the meal pad comprises a test diet containing a predetermined diagnostic amount of said insecticide; and (b) assessing the amount of feces produced by the one or more insects over the predetermined time; wherein the amount of feces produced by the one or more insects is indicative of the presence of an insect(s) resistant to the insecticide.
Also provided herein is a method of identifying a compound that has insecticidal activity against a species of insect, comprising: (a) providing one or more insects from a species; (b) giving the one or more insects access to a meal pad of the invention for a predetermined time, wherein the meal pad has been rehydrated and further wherein the meal pad comprises a test diet containing a test compound to be screened for insecticidal activity; and (c) assessing the amount of feces produced by the one or more insects over the predetermined time; wherein the amount of feces produced by said one or more insects is indicative of the presence of an insect(s) resistant to the insecticide.
The feeding disruption assays described herein may be qualitative (i.e., detect the presence or absence of feces) or quantitative (i.e., count, measure, weight the feces, or the like) in nature. The feeding disruption assays of the invention are suitable for field or laboratory use. In particular preferred embodiments, the methods are used in high throughput methods employing automated imaging devices.
These and other aspects of the invention are set forth in more detail in the description of the invention that follows.